Endolysin CHAP domain-carbosilane metallodendrimer complexes with triple action on Gram-negative bacteria: Membrane destabilization, reactive oxygen species production and peptidoglycan degradation

Bacterial resistance to antibiotics is a significant challenge that is associated with increased morbidity and mortality. Gram-negative bacteria are particularly problematic due to an outer membrane (OM). Current alternatives to antibiotics include antimicrobial peptides or proteins and multifunctional systems such as dendrimers. Antimicrobial proteins such as lysins can degrade the bacterial cell wall, whereas dendrimers can permeabilize the OM, enhancing the activity of endolysins against gram-negative bacteria. In this study, we present a three-stage action of endolysin combined with two different carbosilane (CBS) silver metallodendrimers, in which the periphery is modified with N-heterocyclic carbene (NHC) ligands coordinating a silver atom. The different NHC ligands contained hydrophobic methyl or N-donor pyridyl moieties. The effects of these endolysin/dendrimer combinations are based on OM permeabilization, peptidoglycan degradation, and reactive oxygen species production. The results showed that CBS possess a permeabilization effect (first action), significantly reduced bacterial growth at higher concentrations alone and in the presence of endolysin, increased ROS production (second action), and led to bacterial cell damage (third action). The complex formed between the CHAP domain of endolysin and a CBS silver metallodendrimer, with a triple mechanism of action, may represent an excellent alternative to other antimicrobials with only one resistance mechanism.

细菌对抗生素的耐药性是一项重大挑战，其与发病率升高及死亡率上升密切相关。革兰氏阴性菌（Gram-negative bacteria）因存在外膜（outer membrane, OM）而尤为棘手。当前可供选择的抗生素替代疗法包括抗菌肽、抗菌蛋白以及树枝状大分子（dendrimers）等多功能体系。诸如溶菌素（lysins）类的抗菌蛋白可降解细菌细胞壁，而树枝状大分子则可通透外膜（OM），增强内溶菌素（endolysins）对革兰氏阴性菌的抗菌活性。本研究报道了内溶菌素与两种不同碳硅烷（carbosilane, CBS）银金属树枝状大分子的三阶段协同作用机制：此类树枝状大分子的外围接枝有配位银原子的氮杂环卡宾（N-heterocyclic carbene, NHC）配体，且不同的NHC配体分别带有疏水甲基或氮供体吡啶基基团。该内溶菌素-树枝状大分子复合体系的作用效果基于三大机制：外膜通透、肽聚糖（peptidoglycan）降解以及活性氧（reactive oxygen species, ROS）生成。实验结果表明，碳硅烷银金属树枝状大分子具备外膜通透作用（第一阶段作用）；单独使用且浓度较高时，可显著抑制细菌生长，与内溶菌素联用时效果同样显著，同时可促进活性氧生成（第二阶段作用），最终造成细菌细胞损伤（第三阶段作用）。内溶菌素的CHAP结构域与碳硅烷银金属树枝状大分子形成的复合体系具备三重作用机制，有望成为其他仅具备单一作用机制的抗菌剂的优质替代方案。